Milk is a food source of high nutrition value, which is rich in proteins, vitamins, carbohydrates, calcium and other minerals. Except for being utilized as the nutrition source for newborn babies and infants, it can also be used to prevent osteoporosis in older people, and especially elderly people. Lactose is the major carbohydrate in milk. However, people with lactose intolerance cannot decompose the abundant lactose in milk, which is caused by reduction in the activity of lactase-phlorizin hydrolase. Un-decomposed lactose accumulates in the intestine and induces bacterial fermentation, producing carbon dioxide, hydrogen and methane and resulting in symptoms such as abdominal distension, nausea and diarrhea. Such disadvantages limit the use of milk and dairy products including milk or lactose.
The best strategy to treat patients suffering from lactose intolerance syndrome or to prevent the syndrome, is to avoid the ingestion of dairy products containing large amounts of lactose. However, many people, including lactose-intolerant people, enjoy dairy products containing milk or otherwise containing lactose. Therefore, the development of low-lactose milk and other dairy products containing lactose is crucial. It is now known that α-lactalbumin is an abundant calcium metalloprotein in milk. The binding between α-lactalbumin and galactosyltransferase in the Golgi body can modify the specificity of the galactosyltransferase by forming the lactose synthetase binary complex to synthesize lactose. In order to understand the effect of α-lactalbumin on lactogenesis and the relationship between α-lactalbumin and lactose, Stinnakre et al. utilized gene targeting in embryonic stem cells to produce transgenic mice with heterozygous or homozygous deficiencies in the α-lactalbumin gene. They found that the milk of the transgenic mice with homozygous deficiencies in the α-lactalbumin gene lacked not only α-lactalbumin but also lactose. However, the lack of lactose resulted in highly viscous milk since lactose is an important regulator of osmotic pressure during lactation, and thus the female mice cannot feed their pups smoothly. In the milk of the transgenic mice with heterozygous deficiencies in the α-lactalbumin gene, α-lactalbumin decreased by 40% while lactose decreased by 10-20% (Stinnakre, M. G., Vilotte, J. L., Soulier, S. and Mercier, J. C., 1994. Creation and phenotype analysis of α-lactalbumin-deficient mice. Proc. Natl. Acad. Sci. 91: 6544-6548).
In 1996, L'Huillier et al. created a construct wherein a ribozyme 5 (RZ5) gene is downstream of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) so that the RZ5 gene can be specifically expressed in mammary gland cells. Utilizing gene-transformation technology, transgenic mice highly expressing the RZ5 gene in their mammary glands were developed. RZ5 can specifically degrade α-lactalbumin mRNA to reduce the production of α-lactalbumin and further affects the production of lactose (L'Huillier, P. J., Soulier, S., Stinnakre, M. G., Lepourry, L., Davis, S. R., Mercier, J. C. and Vilotte, J. L., 1996. Efficient and specific ribozyme-mediated reduction of bovine alpha-lactalbumin expression in double transgenic mice. Proc. Natl. Acad. Sci. 93: 6698-6703). According to the studies of Berns and Hauswirth (1979), adeno-associated virus (AAV), which is tissue-specific, uses cells in the tissues of human respiratory and gastrointestinal systems as host cells. Moreover, since AAV is resistant to high temperature and low pH, it is potentially resistant to oral administration (Bern, K. I. and Hauswirth, W. W., 1979. Adeno-associated viruses. Adv. Virus. Res. 25: 407-409). In 1998, During et al. reconstructed AAV into a vector, wherein most of the AAV genes were deleted and only 145 bp of terminal repeats were retained. The resulting AAV vector contains few viral genes so that the possibilities of gene recombination and viral gene expression were reduced to minimum. Then a β-galactosidase gene was constructed onto the AAV vector, and the vector was fed to rats through oral administration. It is found that the β-galactosidase gene was highly expressed at the 6th hour post feeding, and the expression was sustained and stable through 6 months (During, M. J., Xu, R., Young, D., Kaplitt, M. G., Sherwin, R. S. and Leone, P., 1998. Peroral gene therapy of lactose intolerance using an adeno-associated virus vector. Nature Medicine 4: 1131-1135).
Even in view of the aforementioned studies, acceptable low-lactose milk has not been developed. In one aspect, the normal osmotic pressure of lactation is affected. In another aspect, the use of viral vectors has potential danger. Therefore, the development of a new technique for producing low-lactose milk and other dairy products containing lactose is necessary. The present invention satisfies this necessity.